RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation

Abstract

In acute promyelocytic leukaemia (APL), the promyelocytic leukaemia (PML) protein is fused to the retinoic acid receptor α (RAR). This disease can be treated effectively with arsenic, which induces PML modification by small ubiquitin-like modifiers (SUMO) and proteasomal degradation. Here we demonstrate that the RING-domain-containing ubiquitin E3 ligase, RNF4 (also known as SNURF), targets poly-SUMO-modified proteins for degradation mediated by ubiquitin. RNF4 depletion or proteasome inhibition led to accumulation of mixed, polyubiquitinated, poly-SUMO chains. PML protein accumulated in RNF4-depleted cells and was ubiquitinated by RNF4 in a SUMO-dependent fashion in vitro. In the absence of RNF4, arsenic failed to induce degradation of PML and SUMO-modified PML accumulated in the nucleus. These results demonstrate that poly-SUMO chains can act as discrete signals from mono-SUMOylation, in this case targeting a poly-SUMOylated substrate for ubiquitin-mediated proteolysis.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: RNF4 preferentially binds poly-SUMO-2 chains.
Figure 2: Four SIMs in RNF4 are required to bind SUMO polymers.
Figure 3: RNF4 preferentially ubiquitinates poly-SUMO-2 chains.
Figure 4: RNF4 knockdown causes accumulation of SUMO-conjugated PML in ND10 nuclear domains.
Figure 5: Proteasome inhibition causes accumulation of PML, SUMO-1 and ubiquitin covalently associated with SUMO-2 in HeLa cells.
Figure 6: RNF4 ubiquitinates PML only when conjugated by SUMO-2.
Figure 7: Arsenic-mediated degradation of PML is RNF4-dependent.

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. 1

    Hay, R. T. SUMO: a history of modification. Mol. Cell 18, 1–12 (2005).

    CAS  Article  Google Scholar 

  2. 2

    Rodriguez, M. S., Dargemont, C. & Hay, R. T. SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting. J. Biol. Chem. 276, 12654–12659 (2001).

    CAS  Article  Google Scholar 

  3. 3

    Tatham, M. H. et al. Polymeric chains of sumo-2 and sumo-3 are conjugated to protein substrates by sae1/sae2 and ubc9. J. Biol. Chem. 276, 35368–35374 (2001).

    CAS  Article  Google Scholar 

  4. 4

    Matic, I. et al. In vivo identification of human SUMO polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy. Mol. Cell Proteomics 1,132–144 (2007).

    Google Scholar 

  5. 5

    Hecker, C. M., Rabiller, M., Haglund, K., Bayer, P. & Dikic, I. Specification of SUMO1 and SUMO2 interacting motifs. J. Biol. Chem. 23, 16117–16127 (2006).

    Article  Google Scholar 

  6. 6

    Song, J., Durrin, L. K., Wilkinson, T. A., Krontiris, T. G . & Chen, Y. Identification of a SUMO-binding motif that recognizes SUMO-modified proteins. Proc. Natl Acad. Sci. USA 101, 14373–14378 (2004).

    CAS  Article  Google Scholar 

  7. 7

    Prudden, J. et al. SUMO-targeted ubiquitin ligases in genome stability. EMBO J. 26, 4089–101 (2007).

    CAS  Article  Google Scholar 

  8. 8

    Sun, H., Leverson, J. D. & Hunter, T. Conserved function of RNF4 family proteins in eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins. EMBO J. 26, 4102–4112 (2007).

    CAS  Article  Google Scholar 

  9. 9

    Uzunova, K. et al. Ubiquitin-dependent proteolytic control of SUMO conjugates. J. Biol. Chem. 47, 34167–34175 (2007).

    Article  Google Scholar 

  10. 10

    Xie, Y. et al. The yeast HEX3–SLX8 heterodimer is a ubiquitin ligase stimulated by substrate sumoylation. J. Biol. Chem. 47, 34176–34184 (2007).

    Article  Google Scholar 

  11. 11

    Moilanen, A. M. et al. Identification of a novel RING finger protein as a co-regulator in steroid receptor-mediated gene transcription. Mol. Cell Biol. 18, 5128–5139 (1998).

    CAS  Article  Google Scholar 

  12. 12

    Hakli, M., Lorick, K. L., Weissman, A. M., Janne, O. A. & Palvimo, J. J. Transcriptional co-regulator SNURF (RNF4) possesses ubiquitin E3 ligase activity. FEBS Lett. 560, 56–62 (2004).

    CAS  Article  Google Scholar 

  13. 13

    Ong, S. E., Kratchmarova, I. & Mann, M. Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC). J. Proteome Res. 2, 173–181 (2003).

    CAS  Article  Google Scholar 

  14. 14

    Chen, G. Q. et al. In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML–RAR α/PML proteins. Blood 88, 1052–1061 (1996).

    CAS  PubMed  Google Scholar 

  15. 15

    Lallemand-Breitenbach, V. et al. Role of promyelocytic leukemia (PML) sumolation in nuclear body formation, 11S proteasome recruitment, and As2O3-induced PML or PML/retinoic acid receptor alpha degradation. J. Exp. Med. 193, 1361–1371 (2001).

    CAS  Article  Google Scholar 

  16. 16

    Muller, S., Miller, W. H., Jr & Dejean, A. Trivalent antimonials induce degradation of the PML–RAR oncoprotein and reorganization of the promyelocytic leukemia nuclear bodies in acute promyelocytic leukemia NB4 cells. Blood 92, 4308–4316 (1998).

    CAS  PubMed  Google Scholar 

  17. 17

    Shao, W. et al. Arsenic trioxide as an inducer of apoptosis and loss of PML/RAR α protein in acute promyelocytic leukemia cells. J. Natl Cancer Inst. 90, 124–133 (1998).

    CAS  Article  Google Scholar 

  18. 18

    Sternsdorf, T. et al. PIC-1/SUMO-1-modified PML–retinoic acid receptor α mediates arsenic trioxide-induced apoptosis in acute promyelocytic leukemia. Mol. Cell Biol. 19, 5170–5178 (1999).

    CAS  Article  Google Scholar 

  19. 19

    Zhu, J. et al. Arsenic-induced PML targeting onto nuclear bodies: implications for the treatment of acute promyelocytic leukemia. Proc. Natl Acad. Sci. USA 94, 3978–3983 (1997).

    CAS  Article  Google Scholar 

  20. 20

    Duprez, E. et al. SUMO-1 modification of the acute promyelocytic leukaemia protein PML: implications for nuclear localisation. J. Cell Sci. 112, 381–393 (1999).

    CAS  PubMed  Google Scholar 

  21. 21

    Kamitani, T. et al. Identification of three major sentrinization sites in PML. J. Biol. Chem. 273, 26675–26682 (1998).

    CAS  Article  Google Scholar 

  22. 22

    Burgess, R. C., Rahman, S., Lisby, M., Rothstein, R. & Zhao, X. The slx5–slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination. Mol. Cell Biol. 27, 6153–6162 (2007).

    CAS  Article  Google Scholar 

  23. 23

    Kosoy, A., Calonge, T. M., Outwin, E. A. & O'Connell, M. J. Fission yeast Rnf4 homologs are required for DNA repair. J. Biol. Chem. 282, 20388–20394 (2007).

    CAS  Article  Google Scholar 

  24. 24

    Wang, Z., Jones, G. M. & Prelich, G. Genetic analysis connects SLX5 and SLX8 to the SUMO pathway in Saccharomyces cerevisiae. Genetics 172, 1499–1509 (2006).

    CAS  Article  Google Scholar 

  25. 25

    Hakli, M., Karvonen, U., Janne, O. A. & Palvimo, J. J. SUMO-1 promotes association of SNURF (RNF4) with PML nuclear bodies. Exp. Cell Res. 304, 224–233 (2005).

    Article  Google Scholar 

  26. 26

    Brons-Poulsen, J., Petersen, N. E., Horder, M. & Kristiansen, K. An improved PCR-based method for site directed mutagenesis using megaprimers. Mol. Cell Probes 12, 345–348 (1998).

    CAS  Article  Google Scholar 

  27. 27

    Shen, L. et al. SUMO protease SENP1 induces isomerization of the scissile peptide bond. Nature Struct. Mol. Biol. 13, 1069–1077 (2006).

    CAS  Article  Google Scholar 

  28. 28

    Desterro, J. M., Thomson, J. & Hay, R. T. Ubch9 conjugates SUMO but not ubiquitin. FEBS Lett. 417, 297–300 (1997).

    CAS  Article  Google Scholar 

  29. 29

    Shen, L. N., Dong, C., Liu, H., Naismith, J. H. & Hay, R. T. The structure of SENP1–SUMO-2 complex suggests a structural basis for discrimination between SUMO paralogues during processing. Biochem. J. 397, 279–288 (2006).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by Cancer Research UK and the RUBICON EU Network of Excellence. A. P was supported by a Wellcome Trust Studentship. We would like to thank Hugues de The for helpful discussions and providing the chicken anti-PML antibody, and Douglas Lamont, manager of the Fingerprints Proteomics Facility, University of Dundee, for generating the Orbitrap mass spectrometry data. The provision of critical reagents by Roel van Driel and Dan Bailey is gratefully acknowledged.

Author information

Affiliations

Authors

Contributions

M. H. T. carried out the biochemical and proteomic analyses and participated in the writing of the manuscript; M. C. G. carried out the in vivo analysis of PML degradation; L. S. generated and assayed expressed proteins and mutants of RNF4; A. P. carried out ubiquitin site-mapping on poly-SUMO chains modified in vitro; N. H. was involved in initial immunofluorescence microscopy studies; E. J. G. established conditions for siRNA depletion of RNF4; J. J. P. generated expression constructs and antibodies to RNF4; R. T. H. conceived the project and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Ronald T. Hay.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures S1, S2, S3, S4, S5 (PDF 1450 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Tatham, M., Geoffroy, MC., Shen, L. et al. RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation. Nat Cell Biol 10, 538–546 (2008). https://doi.org/10.1038/ncb1716

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing